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In-vitro evaluation of selected Egyptian traditional disease
Ali et al. BMC Complementary and Alternative Medicine 2013, 13:121
http://www.biomedcentral.com/1472-6882/13/121
RESEARCH ARTICLE
Open Access
In-vitro evaluation of selected Egyptian traditional
herbal medicines for treatment of alzheimer
disease
Shereen K Ali1, Ahmed R Hamed1,2, Maha M Soltan1,2, Usama M Hegazy3, Esameldin E Elgorashi4, Ibrahim A El-Garf5
and Ahmed A Hussein6*
Abstract
Background: Egyptians recognized the healing power of herbs and used them in their medicinal formulations.
Nowadays, “Attarin” drug shops and the public use mainly the Unani medicinal system for treatment of their health
problems including improvement of memory and old age related diseases. Numerous medicinal plants have been
described in old literature of Arabic traditional medicine for treatment of Alzheimer’s disease (AD) (or to strengthen
memory).
Methods: In this study, some of these plants were evaluated against three different preliminary bioassays related to
AD to explore the possible way of their bio-interaction. Twenty three selected plants were extracted with methanol
and screened in vitro against acetylcholinesterase (AChE) and cycloxygenase-1 (COX-1) enzymes. In addition,
anti-oxidant activity using DPPH was determined.
Results: Of the tested plant extracts; Adhatoda vasica and Peganum harmala showed inhibitory effect on AChE
at IC50 294 μg/ml and 68 μg/ml respectively. Moreover, A. vasica interacted reversibly with the enzyme while
P. harmala showed irreversible inhibition. Ferula assafoetida (IC50 3.2 μg/ml), Syzygium aromaticum (34.9 μg/ml)
and Zingiber officinalis (33.6 μg/ml) showed activity against COX-1 enzyme. Potent radical scavenging activity was
demonstrated by three plant extracts Terminalia chebula (EC50 2.2 μg/ml), T. arjuna (3.1 μg/ml) and Emblica officinalis
(6.3 μg/ml).
Conclusion: Interestingly, differential results have been obtained which indicate the variability of the mode of
actions for the selected plants. Additionally, the reversible interaction of A. vasica against AChE and the potent
activity of F. assafoetida against COX-1 make them effective, new and promising agents for treatment of AD in the
future, either as total extracts or their single bioactive constituents.
Keywords: Egyptian herbal medicine, Unani medicine, Alzheimer’s disease, Anti-acetylcholinesterase,
Anti-inflammatory, Anti-oxidant, Adhatoda vasica, Ferula assafoetida
Background
Alzheimer’s disease (AD) is a neurodegenerative disorder
characterized by a progressive decline of memory and
cognition. Amyloid-β (Ab), neurofibrillary tangles (NFT)
and synaptic loss; particularly, the deficiency of acetylcholine (ACh) and the degeneration of cholinergic neurons in the cortex and hippocampus, nucleus basalis of
* Correspondence: [email protected]
6
Chemistry Department, University of Western Cape, Private Bag X17,
Belleville 7535, South Africa
Full list of author information is available at the end of the article
Meynert, are the hallmarks of AD [1,2]. A loss of ACh is
considered to play a vital role in the learning and memory deterioration of AD patients. Acetylcholine is an
organic molecule liberated at nerve endings as a neurotransmitter. It is produced by the synthetic enzyme choline acetyltransferase which uses acetyl coenzyme-A and
choline as substrates for the formation of acetylcholine
in specific cells known as cholinergic neurons. Neurotransmitter disturbances and insufficient cholinergic
functions are identified among the pathological features
in central nervous system disorders [3].
© 2013 Ali et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Ali et al. BMC Complementary and Alternative Medicine 2013, 13:121
http://www.biomedcentral.com/1472-6882/13/121
Because of the complication of ACh deficiency in AD
patients, elevating ACh level is an essential target for
treatment. There are many strategies that can be used
to enhance ACh level such as using ACh precursor
(choline) [4], muscarinic and nicotinic agonists [5],
ACh releasers [6] and AChE inhibitors [7]. However
AChE inhibitors have some complications such as toxicity or resistance by increasing AChE expression level
[8], but use of AChE inhibitors in AD patients has been
the most effective strategy up to date.
Inflammation is a disorder involving localized increase
in the number of leukocytes and a variety of complex
mediator molecules. Prostaglandins are ubiquitous substances that initiate and modulate cell and tissue responses involved in inflammation. Their biosynthesis
has also been implicated in the pathophysiology of cardiovascular diseases, cancer, colonic adenomas and
Alzheimer’s disease [9].
Oxidative stress refers to the physiological condition at
which the capacity of the endogenous antioxidant system
fails to cope with the damaging effects of free radicals.
Strong experimental evidences have been established
about the oxidative stress theory of AD pathogenesis
where oxidative damage plays a major role in neurological degeneration [10].
The ancient Egyptians had a system of medicine that
was very advanced for its time and influenced later medical traditions. When the Arabs came to Egypt, Arabic
medicine was practiced and the art of healing made use
of all available knowledge gained from different civilizations such as the Persian, Chinese, Greek, as well as the
Ancient Egyptian. The books written by some famous
scholars such as Al-antaki [11], Al-turkimany [12], Ibn
Sina [13], and Ibn el-Bitar [14] which represent the main
references in herbal shops (known as Attarin), described
in their books a number of conditions related to AD and
recommended numerous herbal medicine to improve
the old ageing problems including AD. In this study we
analysed these books and abstracted the information on
plants described for the treatment of old age diseases
like Alzheimer, joint inflammations …etc.
Currently, a few drugs are available in the market as
safe and effective for the treatment of AD. Thus, in this
article we evaluated in vitro some Egyptian herbal medicines that have been highly recommended in old Arabic
literature for treatment of AD to discover newly potent
and safe natural therapeutic agents for treatment of AD.
Methods
Page 2 of 10
believed to be the main references used in the “Attarin”
shops in Cairo. In this study we reviewed the information
given by some scholars like Dawood el Antaki [11], Altorkmany [12], Ibn Sina [13], and Ibn el-Bitar [14].
Plant materials (leaves, roots and seeds) were collected
from either their natural habitats or the local market
(Table 1). Two plants Boswellia scara (supplied and
identified by Dr. A. al_Adawi, Ghadafan Agriculture
Research Station, Ministry of Agriculture and Fisheries,
Sohar, Sultanate of Oman), and Ferula assafoetida (supplied by Dr. M. Ziaratnia, Research Institute of Food
Science and Technology, Isfahan, Iran). Voucher specimens (Table 1) were identified by Prof. Ibrahim El-garf,
a co-author of this article, and deposited in the Department of Phytochemistry, National Research Centre,
Egypt. The collected fresh materials were dried, powdered and extracted by homogenization with methanol
(10 ml g−1), using electrical blender and macerated
overnight then filtrated, the residues were re-extracted
three times with fresh solvent. The filtrates were combined and the solvent removed at 45°C under reduced
pressure. The total extracts were kept at ~ −5°C for further use.
The multi-well plate AChE inhibition assay
The AChE inhibitory activity of each extract was tested
using 96 well micro-plate assay based on previously
published methods [15,16] with minor modifications.
Each extract (25 μl of 10× of final concentrations in
DMSO) was dispensed in duplicates onto 96 well microplate and mixed with 200 μl of Ellman’s mixture
containing 10 mM Tris–HCl, pH 8, 0.1% bovine serum
albumin (BSA, fraction V), 1.5 mM acetylthiocholine
iodide (ATCI, Sigma-Aldrich, Germany) and 3 mM 5,5'dithio-bis-(2-nitrobenzoic acid) (DTNB, Sigma-Aldrich,
Germany). The control wells contained the extract vehicle (DMSO) instead of the extract. The reaction was
started with the addition of enzyme solution (25 μl, 0.1
U/ml). Autohydrolysis of the substrate was corrected by
replacing the enzyme with 25 μl of enzyme buffer
(10 mM Tris–HCl, pH 8, containing 0.1% BSA) in duplicate wells. The enzymatic activity was monitored kinetically at 450 nm every 30 s intervals for 3 min at 30°C
(linear reaction). The enzyme rate was calculated from
the slope of the curve of absorbance change vs time. As
screening strategy, final concentration of 1000 μg/ml
from each extract was examined and the average % inhibition was calculated relative to the enzyme rate at
the vehicle control wells according to equation 1:
Plant materials
Selection of plant species screened in this study was
based on their uses in Egyptian traditional medicine
(Table 1). Information was gleaned from different
sources of old Arabic literature available which are
% Inhibition ¼ mean slopes of the vehicle control−mean slopes of the sample
mean slopes of the vehicle control
Equation 1. Calculation of the average % inhibition of
different extracts on AChE.
Ali et al. BMC Complementary and Alternative Medicine 2013, 13:121
http://www.biomedcentral.com/1472-6882/13/121
Page 3 of 10
Table 1 Egyptian herbal medicines reported for treatment of age- related diseases
No. Plant species
Plant family
Voucher Part
No.
used
Collection
site
Arabic
name
Traditional uses
1
Adhatoda vasica Nees.
Acanthaceae
STDF-1
Aerial
parts
El-Orman
garden
No traditional use reported
2
Aloe vera. L.
Aloaceae
STDF-2
Dried
juice
El-Orman
garden
Improves mental capacity, benefits vitality,
anti-depressant [12]. Used by pharaohs for chest
pains, headaches, skin diseases and allergies [21],
3
Anacyclus pyrethrum L.
Asteraceae
STDF-5
Roots
Herbal shop
Nerve tonic, improves cerebral blood
circulation, remedy for paralysis [12].
4
Boswellia sacra Flueck.
Burseraceae
STDF-6
Gum
Oman
Anti-depressant enhances mental capacity,
cures frequent forgetfulness, [12,14]. Boswellia sp.
was used by ancient Egyptians for rheumatism,
joint pain and facial wrinkles [21].
5
Brassica rapa ssp rapa
Brassicaceae
STDF-8
Root
Local market
Aphrodisiac, anti-ageing, hearing disorders [12].
6
Brassica nigra L.
Brassicaceae
STDF-9
Seeds
Herbal shop
Anti-ageing strengthens the vitality [21].
Aphrodisiac, joints disorders and chest pain
[11,13,14]. The Pharaohs used mustard seeds to
treat muscle, joint and chest pains [21].
7
Emblica officinalis Gaertn
Euphorbiaceae
STDF-10
Fruits
El-Fayom
Improves memory, stimulant, and restoratives
for all organs [21].
8
Ferula assafoetida Boiss.
& Buhse
Apiaceae
STDF-12
Gum
Iran
Stimulant, strong aphrodisiac, strong nerve tonic,
relieves on-going mental and physical fatigue,
joints inflammation, depression and sadness [12].
Treat weakness of sexual desire and nerves [11].
9
Melilotus officinalis (L.) Pall. Fabaceae
STDF-13
Aerial
parts
Orman
garden
Joints pains [12].
10
Cassia fistula L.
Fabaceae
STDF-14
Fruits
Orman
garden
Tonic, detoxicant [12]. expectorant for brain and
chest problems [11]. Relieves inflammations of
nerves and joints [13,14].
11
Nerium oleander L.
Apocynaceae
STDF-15
Leaves
Orman
garden
Highly toxic, relieves knee and back pain [12].
12
Nigella sativa L.
Ranunculaceae
STDF-16
Seeds
Herbal shop
Stimulant, improving memory, resolutive,
considered as an adaptogen [12].
13
Peganum harmala L.
Zygophyllaceae STDF-21
Seeds
South Sinai
Hallucinogenic, epilepsy, mental and nervous
illnesses, relieves joints inflammation [12].
Cures headaches, strokes, numbness, epilepsy
and forgetfulness [11].
14
Piper nigrum L.
Piperaceae
STDF-22
Seeds
Herbal shop
Stimulant, memory enhancer, sharpens the mind,
and for strokes [11,12]. Piper sp. (Piper cubeba)
used by Pharaohs against different types of
infections and headaches [21].
15
Rheum palmatum L.
Polygonaceae
STDF-23
Stem
Herbal shop
Anti-ageing, for dyspepsia, improves memory,
and maintains healthy mind [12].
16
Rosmarinus officinalis L.
Lamiaceae
STDF-24
Aerial
parts
Orman
garden
Sharpens the mind, anti-depressant, anxiety,
poor memory, and rheumatoid arthritis.
17
Ruta graveolens L.
Rutaceae
STDF-25
Leaves
Local market
Memory enhancer, relieves strokes, tremors,
convulsion and epilepsy, joint pains [12].
18
Salvia triloba L.
Lamiaceae
STDF-26
Aerial
parts
Sinai
(El-A’rish)
Anti-inflammatory, nerve tonic, and
memory enhancer.
19
Syzygium aromaticum (L.)
Merrill & Perry
Myrtaceae
STDF-27
Pud
Herbal shop
General tonic and memory enhancer [12].
Stimulant for brain, and anti-depressant [11].
Ali et al. BMC Complementary and Alternative Medicine 2013, 13:121
http://www.biomedcentral.com/1472-6882/13/121
Page 4 of 10
Table 1 Egyptian herbal medicines reported for treatment of age- related diseases (Continued)
20
Terminalia arjuna
(Roxb.) Wight & Arn.
Combretaceae
STDF-28
Fruits
El-Fayom
Highly recommended for ageing diseases,
improves memory and brain function, keeps
the brain young and healthy [12]. Strengthens the
senses and the brain, improves memory [11,13].
21
Terminalia chebula Retz.
Combretaceae
STDF-29
Fruits
El-Giza
Traditionally used like T. arjuna.
22
Teucrium polium L.
Lamiaceae
STDF-30
Aerial
parts
South Sinai
Improves mental performance, and
concentration [12].
23
Zingiber officinale Roscoe
Zingiberaceae
STDF-31
Rhizome Local market
(Mepaco)
Five serial dilutions were prepared from the extracts
that showed more than 50% inhibition to determine the
IC50 (extract concentration producing 50% inhibition of
AChE activity as generated by non-linear regression analysis). Galanthamine (Sigma-Aldrich, Germany) served
as positive control.
Determination of the inhibition type of plant extracts
on AChE
The type of inhibition of AChE by P. harmala and A.
vasica extracts (reversible or irreversible inhibition) was
determined by measuring the restored AChE activity by
10 time dilution of plant extract concentration after
mixing and incubation of AChE and plant extract. AChE
activity was measured after gentle mixing of 110 μl of
(100 μl enzyme:10 μl plant extract) with 890 μl of mixture containing 10 mM Tris–HCl, pH 8, 0.1% BSA,
1.5 mM ATCI, 3 mM DTNB and 90 μl plant extract. In
a separate experiment, the dilution effect of plant extract
on AChE activity was measured after gentle mixing
110 μl of (100 μl enzyme:10 μl plant extract) with 890 μl
of the same above mixture except that 90 μl plant
extract was replaced with 90 μl DMSO (solvent). In reversible inhibition, AChE activity can be restored by dilution of plant extract, while there is no change in AChE
activity with dilution of plant extract in irreversible
inhibition.
Cyclooxygenase-1 assay
Inhibition of prostaglandin biosynthesis by the plant
extracts was investigated using COX-1 assay [17]. Indomethacin was included as a standard. Per cent inhibition of plant extracts was calculated by comparing the
amount of radioactivity present in the sample with that
in the solvent blank.
Antioxidant activity: 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical scavenging assay
Plant extracts/compounds were prepared in DMSO as
10× stocks from each test concentration (between 0–
100 μg/ml) and briefly sonicated when necessary in an
ultrasonic water bath. Plant extracts/compounds producing
Memory enhancer, for joints inflammation [11-13].
radical scavenging activities equal to or higher than 50% at
100 μg/ml in a preliminary screen were further tested and
EC50 (concentration of the extract/compound producing
50% scavenging of DPPH radicals) determined using nonlinear regression analysis of the dose-%AA relationship
(Equation 1). Three reference radical scavengers (quercetin,
gallic acid and t-butylhydroquinone) were tested in the
assay as positive controls. The assay method used in the
present study was based on a modified procedure [18]
which is based essentially on previously published literature [19]. The plant extract/compound stock solutions
(20 μl/well) were dispensed in duplicates onto 96-well
plates (flat-bottomed, Greiner bio one, Belgium). The
assay was started with the addition of DPPH reagent
(0.004% wt/v in methanol, 180 μl/well). Appropriate
blanks were prepared using the solvent only in addition to
the same amount of DPPH reagent to get rid of any inherent solvent activity. Negative controls were also run in
parallel to correct for any non-DPPH absorbance by
coloured extracts at the test wavelength. The plate was
immediately shaken for 30 seconds and incubated in the
dark for 30 minutes at room temperature. The remaining
DPPH was measured in the microplate reader at 540 nm.
The percentage of antioxidant activity (%AA) was calculated according to equation 2:
% Antioxidant activity DPPH ð%AAÞ ¼ 100
½OD540ðblank Þ−OD540 ðsampleÞ
OD540 ðblank Þ
Equation 2. Calculation of the % AA for DPPH assay.
OD540 (blank) and OD540 (sample) are the averages of
duplicate determinations of the corrected readings of
blank and sample at 540 nm, respectively.
Results
The plants (Table 1) were selected based on their traditional uses for treatment of AD or age related diseases
except for A. vasica which was selected on the basis of
chemotaxonomy. The plants were collected from their
natural habitats or from the “Attarin” or the herb shops.
A small portion of plant parts (100 g) were extracted
Ali et al. BMC Complementary and Alternative Medicine 2013, 13:121
http://www.biomedcentral.com/1472-6882/13/121
Page 5 of 10
Table 2 Biological activities of Egyptian herbal medicines against different bioassays related to AD
No.
Plant species
Screening data
IC50 (μg/ml)
% inhibition of ACHEa
% inhibition of COX-1b
DPPH (%AA) b
ACHE
COX-1
DPPH
86.0
-
19.0
294
N.D
>100
1
A.vasica
2
A. vera.
-
-
31.0
-
N.D
>100
3
A. pyrethrum L.
21.0
-
90.0
N.D
N.D
26.3
4
B. sacra
−46.0
15.1 ± 5.7
8.0
N.D
N.D
>100
5
B. alba
16.0
52.3 ± 2.1
24.0
N.D
N.D
>100
6
B. nigra.
19.0
10.9 ± 1.6
42.0
N.D
N.D
>100
7
E. officinalis
-
-
97.0
N.D
N.D
6.3
8
F. assafoetida
3.0
96.7 ± 1.3
17.0
N.D
3.2
>100
9
M. officinalis
−49.0
-
6.0
N.D
N.D
>100
10
C. fistula.
−16.0
19.2 ± 4.8
66.0
N.D
N.D
75.0
11
N. oleander.
-
-
60.0
N.D
N.D
64.5
12
N. sativa.
5.0
55.4 ± 8.8
19.0
N.D
N.D
>100
13
P. harmala.
92.0
-
41.0
68
N.D
>100
14
P. nigrum.
34.0
-
30.0
N.D
N.D
>100
15
R. palmatum.
−20.0
-
95.0
N.D
N.D
14.2
16
R. officinalis.
10.0
11 ± 5.4
82.0
N.D
N.D
19.4
17
R. graveolens.
8.0
-
69.0
N.D
N.D
61.0
18
S. triloba.
-
-
93.0
N.D
N.D
20.7
19
S. aromaticum
47.0
80.3 ± 0.9
89.0
N.D
34.9
15.9
20
T. arjuna
−10.0
-
96.0
N.D
N.D
3.1
21
T. chebula
13.0
-
95.0
N.D
N.D
2.2
22
T. polium
16.0
-
51.0
N.D
N.D
96.4
23
Z. officinalis
−45.0
68.2 ± 3.1
47.0
N.D
33.6
>100
Galanthamine
n/a
n/a
n/a
9.4
n/a
n/a
Indomethancin
n/a
n/a
n/a
n/a
0.61
n/a
Reference DPPH scavengers:
t-BHQ
2.8
Gallic acid
1.2
Quercetin
4.5
at 1000 μg/ml, at 100 μg/ml, N.D not detected, n/a not applicable.
a
b
and tested in different in-vitro bioassays related to
AD. The medicinal uses of the listed plants (Table 1)
were discussed in detail by numerous scholars as Ibn
Sina “Avicenna”, Ibn El Beitar, El Baironi, Al Antaki,
Al Mo’tamed [11-14,20].
The inhibition effect of the methanolic extracts from
the 23 different extracts on AChE activity was screened
(Table 2, Figure 1). The screening was performed at a
concentration of 1000 μg/ml and the activity guidelines
of our program only considered the extracts as active if
they only inhibited the enzyme more than 50%. The
screening showed different effects on AChE activity as
shown in Table 2. Extracts from M. officinalis, B. sacra,
and Z. officinalis activated AChE more than 45%. Only,
two species namely A. vasica and P. harmala inhibited
AChE by 86 and 90% respectively (Figure 1). Further
testing and analyses of the inhibition of AChE by A.
vasica and P. harmala revealed the IC50 values of 294
and 68 μg/ml respectively.
The inhibition type of A. vasica and P. harmala was determined by assaying the change in the remaining AChE
activity of the mixture of AChE and the plant extract before and after the dilution of the plant extract in the same
mixture, while, AChE activity increased 5 fold by 10 times
dilution of A. vasica, the same dilution of P. harmala did
not show any effect on the remaining activity of AChE
after dilution. This result indicates that AChE is inhibited
reversibly by A. vasica and irreversibly by P. harmala.
Screening of the extracts against COX-1 enzyme at
100 μg/ml showed that six extracts demonstrated more
Ali et al. BMC Complementary and Alternative Medicine 2013, 13:121
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100
% AChE inhibition
80
60
40
20
0
-20
-40
-60
1 3 4 5 6 8 9 10 12 13 14 15 16 19 17 20 21 22 23
Plant No.
Figure 1 % inhibition of AChE by different plant extracts at
1000 μg/ml. Plant No. on x-axis refers to the corresponding
numbered plants in Table 1.
than 50% inhibition (Table 2). The IC50 of the active extract
showed potent inhibitory activity for F. assafoetida (IC50
3.2 μg/ml) and moderate activity for Z. officinalis (33.6 μg/ml).
Table 2 shows the anti-oxidant results of the tested
plant extracts, three of them; T. chebula (EC50 2.2 μg/
ml), T. arjuna (3.1 μg/ml) and E. officinalis (6.3 μg/ml)
were particularly strong antioxidants when compared to
the reference radical scavengers (t-BHQ, gallic acid and
quercetin) recording EC50’s < 10 μg/ml. Five species
showed the activity at EC50’s of 10–30 μg/ml; these were
R. palmatum (EC50 14.2 μg/ml), S. aromaticum (15.9 μg/
ml), R. officinalis (19.4 μg/ml), S. tribula (20.7 μg/ml)
and A. pyrethrum (26.3 μg/ml). Another four species R.
graveolens, N. oleander, C. fistula and T. polium showed
the activity at EC50 of 30–100 μg/ml. The rest of the
examined plant species either showed weak activity or
were inactive at 100 μg/ml.
Discussion
Ancient Egyptians were familiar with drug preparation
from plants and herbs such as cumin, fennel, caraway,
aloe, safflower, pomegranates, and castor and linseed oils
[21]. However, nowadays, the majority of the herbal
medicine information is coming from Unani medicine,
some of the plant still originated from pharaonic era,
and still used for treatment of different diseases like
Boswellia sp., aloe, and mustard.
The deficiency of ACh is one of characteristics of AD
and responsible for most of the AD symptoms such as
decline of memory and cognition of the AD’s patients.
AChE inhibitors such as tacrine, donepezil, rivastigmine,
and galanthamine are effective anti-AD drugs in the
market [22]. The side effects of anti-AChE drugs such as
toxicity, tolerability, and loss of efficiency stimulates the
researchers to screen alternative natural anti-AD drugs
for medication switch [23].
In the present work, the selected extracts were
screened for AChE inhibition. Only A. vasica and P.
Page 6 of 10
harmala showed inhibitory activity against AChE with
IC50 value 294 and 68 μg/ml, respectively. Both plants
contain β-carboline alkaloids, which demonstrated
potent activity against AChE [24]. Extracts from natural
resources usually containing un-determined number of
secondary metabolites and expected to play different role
upon their interaction with human biological system. In
this study the major biological activity demonstrated by
both extracts could be attributed to the dominant major
constituents in each extracts which should be able to go
through blood brain barrier and interact with the active
sites. The major constituent of P. harmala is harmaline
and A. vescia contains vasicine (from 0.0541 to 1.105%).
Generally β-carbolines are a large group of natural indole
alkaloids that are widely distributed in nature. They
possess diverse pharmacological activities such as sedative, hypnotic, anxiolytic, anticonvulsant, antitumor,
antithrombotic, antiparasitic, antimicrobial, as well as antiviral activities [24]. Harmaline, the major active constituent of P. harmala is a common dihydro β-carboline type,
it possess interesting pharmacological activities and can
interact with several enzymes and neurotransmittors
including topoisomerase I, and monoamine oxidase-A
[25,26]. The different activities demonstrated by both
major compounds could explain the difference in relative potential IC50 values of both plants. Although, P.
harmala has been used in traditional medicine, there
are reports of severe intoxication in cattle, donkeys, sheep
and horses [27]. Digestive and nervous syndromes have
been reported in animals that consume a sub-lethal
amount of the plant. Harmaline and harmine are toxic
alkaloids characterized in the seeds of P. harmala.
Harmaline is almost twice as toxic as harmine and in
moderate doses cause tremors and clonic convulsions,
but with no increase in spinal reflex excitability [28]. A
vasica showed safety when intragastrically administrated at 2.5 g/kg, clinical trials performed on combination preparations containing A. vasica showed no
serious adverse effects [29].
Although IC50 value of P. harmala is about four times
higher than that of A. vasica, the inhibition type study
showed that A. vasica reversibly inhibits AChE and can
be used for AD’s medication rather than P. harmala
which inhibits irreversibly AChE. This recommendation
was supported by the toxicity reports in literature which
indicated the higher safety margin of A. vasica as compared to P. harmala.
According to the best of our knowledge this is the first
report about the reversible anti-chloinsterase interaction
of A vasica extracts growing in Egypt, which add new
value and activity to this important plant.
Anti-inflammatory COX-inhibiting NSAIDs have received increased attention in experimental and therapeutic trials for Alzheimer’s disease. Interestingly,
Ali et al. BMC Complementary and Alternative Medicine 2013, 13:121
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COX-1-expressing microglia surrounds amyloid plaques.
There is no evidence that COX-1 expression in microglia
is changed in AD brain. However, accumulation of COX1-expressing microglia in AD could result in local increase
in prostaglandin synthesis and oxidative stress. F.
assafeotida demonstrated potent inhibitory activity against
COX-1 enzyme. Asafoetida used in traditional medicine to
improve memory and as an antihelminthic, antispasmodic
and antibacterial agent [11-14]. Z. officinalis showed potent inhibitory activity against COX-1 enzyme, and also,
demonstrated high radical scavenging properties, which
may attributed to their contents of gingerols and
shoagols. Further understanding of the role of COX inhibitory activity of herbal medicine in mechanisms leading to AD generation is critical to the future development
of NSAID therapy for AD from traditional medicine [30].
Increased oxidative stress causes cell damage in the
form of protein, lipid, and DNA oxidations. Elevated
ROS levels are also associated with increased deposition
of amyloid- and formation of senile plaques, a hallmark
of the AD brain. If enhanced ROS exceeds the basal level
of cellular protective mechanisms, oxidative damage and
cell death will result. Therefore, the plant extracts which
demonstrated potent free radical scavenging properties
particularly those showed EC50 < 10 μg/ml (T. chebula,
T. arjuna and E. officinalis) expected to play a vital role
in reducing the oxidative stress and this may explain
their use in traditional medicine for improvement of AD
and/or ageing related diseases.
Brassica was reported to be used traditionally against
many human diseases including AD. It contains potential bioactive phytochemicals. Isothiocyanate derivatives
from Brassicaceae increased NGF-induced neurite elongation by ∼ 70%. It’s also induced sustained production of
β-tubulin in the presence of NGF enhancers [31]. Plant
sterols including brassicasterol are solely dietary-derivable
sterols that are structurally very similar to cholesterol and
can cross the blood–brain barrier and accumulate within
mammalian brain and may play an important role in
protection against AD [32]. Sinapic acid showed antiinflammatory and neuroprotective activities, the mechanism of action involve amelioration of Aβ(1–42)
protein-related pathology including neuronal cell death
and cognitive dysfunction [33]. Sinapine is another compound which is widely spread in Brasicaceae, significantly
inhibited AChE activity on cerebral homogenate (IC50
3.66 μmol/L-1) [34].
Cassia fistula native to southern Asia, and widely distributed in Egypt as an ornamental tree. The seeds from
the fruit are well known in Unani traditional medicine
and widely used for medicinal purposes. It was described
as safe and efficient purgative even for pregnant women
and for children. Recently, the effects of the seed extracts against ageing diseases have been documented.
Page 7 of 10
The ethanolic extract of the seeds of C. obtusifolia (synonym C. fistula) (COE), significantly attenuates memory
impairment induced by scopolamine via acetylcholinesterase inhibition [35]. COE attenuated secondary Ca2+
dysregulation induced by NMDA (700 μM), while a preapplication of COE reduced NMDA-induced cell death.
Furthermore, COE was neuroprotective against the mitochondrial toxin 3-NP (1 mM) [36]. Some of the isolated
compounds were shown to inhibit the activities of βsecretase and enhance the memory in the animals with
scopolamine-induced memory loss [37].
Emblica officinalis (Amla) grows in tropical and subtropical parts of East Asia, and was cultivated in Egypt
in the last few years for its economic value. In traditional
medicine, E. officinalis is used for various conditions like
diarrhea, jaundice, inflammation, cerebral insufficiency
and mental disorders [38]. It is used as a tonic for heart
and brain in Unani medicine. The extract demonstrated
various pharmacological activities. Amla churna (powdered dry fruit of amla) has also been reported to produce a dose-dependent improvement in memory scores
of young and aged rats [39,40]. E. officinalis extract has
an ability to improve or ameliorate spatial long-term
memory and short-term memory attributable to mechanisms like antioxidant, anti-inflammatory, AChE inhibitory, hypolipidemic and neuroprotective activities [41].
Nerium oleander (oleander) belongs to the family Apocynaceae. It is widely cultivated as a garden plant, which
showed interesting anticancer activity. Unani system
recommended the topical uses of the plant more than
the internal use, which should be administrated under
supervision and with caution. The anti-ageing properties
of the plant extract was documented recently, the polysaccharides isolated from the flowers of oleander showed
potential neuroprotective activity against neuronal death
in Alzheimer’s disease and the neuroprotective mechanism may primarily rely on inactivation of JNK signaling pathway [42,43]. Also, cardiac glycoside derivatives
are proposed as treatment for Alzheimer’s disease,
Huntington’s disease or stroke [44].
Nigella sativa is considered as an adaprogenic herb
and is widely used in Egypt and other Arabic countries;
it showed no activity in vitro against cholinesterase
[45], but in vivo, the fixed oil has demonstrated noticeable spatial cognitive preservation in rats challenged
with chronic cerebral hypoperfusion which indicates a
promising prospective neuroprotective effect [46].
Ruta graveolens (common rue) is cultivated in many
parts of the world; it has been used for centuries as a
medical preparation. In Unani system it is used as stimulant, emmenagogue, diuretic, abortefacient, resolvent
and brain tonic [47]. Methanolic and hexane extracts of
R. graveolens showed potent inhibition of AChE and
butyryl cholinesterase (BuChE) in-vitro [48]. Rue
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contains rutin, which, widely used as a drug to improve
blood circulation and expected to contribute for such
activities.
Sage is a common name for Salvia species, and highly
appreciated over all the world, it is used for treatment of
many diseases and also proved to have strong activity
against AD, in Egypt S. triloba is called Maramaria and
it is used as condiment and tea. The plant has been
reported in old Arabic literature to improve the mental
power [49,50].
Black pepper (Piper nigrum) is a flowering vine in the
family Piperaceae [51]. The plant has been used effectively
for the treatment of AD. Piperine is a major plant alkaloid
present in black pepper (Piper nigrum) and long pepper
(Piper longum), which are among the most common spices
consumed by a large number of people worldwide. This
plant is known to possess several pharmacological actions,
such as antimicrobial, antifungal, anti-inflammatory and
antioxidant effects [52]. Piperine demonstrated in in vitro
studies to protect against oxidative damage by inhibiting
or quenching free radicals and ROS, lower lipid peroxidation in vivo and beneficially influence cellular thiol status,
antioxidant molecules and antioxidant enzymes in a number of experimental situations of oxidative stress [53].
A recent in vivo work conducted by our group, revealed
a significant reduction of the oxidative stress status and
amelioration of the neurodegeneration characteristic of
Alzheimer’s diseases in rats using P. nigrum and S. triloba.
It is noteworthy that S. triloba extract showed more interest in improvement of AD in rats [54].
Terminalia species belong to the family combretaceae.
They are extensively used in Unani, Ayurveda and homeopathic medicine. T. chebula is a popular traditional medicine in many countries including Egypt. It has a wide
spectrum of pharmacological activities and reported as antioxidant, antidiabetic, antibacterial, antiviral, antifungal …
etc. According to Unani medicine, emulsifying of one fruit
every day prevents ageing and keeps the person very
healthy. Recent literature supported the anti-ageing properties of Terminalia species. Phenolic constituents from T.
chebula showed strong AChE and BChE inhibitory activities, and antioxidant activity [55]. T. chebula has been
recommended for old age diseases [56]. Oral administration
of different doses of aqueous extract of T. arjuna causes
significant elevation in activities of catalase, superoxide
dismutase and glutathione S transferase. Also, T. arjuna is
found to down regulate anaerobic metabolites by inhibiting
the activity of lactate dehydrogenase in lymphoma bearing
mice. The strong antioxidant action of aqueous extract of
T. arjuna may play a role in treatment of age-related diseases such as cancer and coronary heart disease and neurodegenerative disorders [57].
The examined biological properties; anti-AChE, antioxidant and anti-inflammatory of the selected species
Page 8 of 10
revealed a diversity of the active species suggesting a
different mechanisms. Additionally, animal based in vivo
research during the last ten years revealed interesting
activities for the majority of the plants listed in Table 1
as discussed above, which, justify their use in traditional
medicine to improve memory or treatment of ageing
diseases including AD by traditional practitioner overall
the world including Egypt.
Conclusion
The reputed medicinal properties of plants have been documented for centuries in different cultures including
Egypt, and there are many plant species that have been
traditionally used for memory disorders as listed in Table 1.
Different results have been obtained which indicate the
variability of the mode of actions for the selected plants.
Additionally, the reversible interaction of A. vasica against
AChE and the potent activity F. assafoetida against COX1 making them effective, new and promising agents for
treatment of AD in the future, either as total extracts or
their single bioactive constituents.
Competing interests
The authors declare that they have no competing interest.
Authors’ contributions
SKA and AAH carried out the collection and extraction of plant materials and
drafting the manuscript. ARH, MMS, UMH participated in acetylcholinesterase,
DPPH, inhibition type bioassays, analysis and interpretation of data and in
the drafted the manuscript. EEE performed COX-1 bioassay. IAE-G identified
the plant materials. All authors read and approved the final manuscript.
Acknowledgement
This work was supported by Science and Technology Development Fund
(STDF), Egypt (project number 251). The authors are grateful to Dr. A.
al_Adawi, Ghadafan Agriculture Research Station, Ministry of Agriculture and
Fisheries, Sohar, Sultanate of Oman, and Dr. M. Ziaratnia, Research Institute of
Food Science and Technology, Isfahan, Iran, for supplying B. sacra and
F. assafoetida plant materials.
Author details
1
Department of Phytochemistry, National Research Centre, 13211 Dokki,
Cairo, Egypt. 2Pharmaceutical Research Group, Centre of Excellence for
Advanced Sciences, National Research Centre, 13211 Dokki, Cairo, Egypt.
3
Molecular Biology Department, Genetic Engineering and Biotechnology
Division, National Research Centre, 13211 Dokki, Cairo, Egypt.
4
Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of
Veterinary Science, University of Pretoria, Onderstepoort 0110, Pretoria, South
Africa. 5Department of Botany, Faculty of Science, Cairo University, El-Giza,
Egypt. 6Chemistry Department, University of Western Cape, Private Bag X17,
Belleville 7535, South Africa.
Received: 3 January 2013 Accepted: 20 May 2013
Published: 30 May 2013
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doi:10.1186/1472-6882-13-121
Cite this article as: Ali et al.: In-vitro evaluation of selected Egyptian
traditional herbal medicines for treatment of alzheimer disease. BMC
Complementary and Alternative Medicine 2013 13:121.
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